This invention relates to a method for the separation of glycolipids from a lipid mixture containing complex lipids (CLS).
Complex lipids are chemical structures that are widespread in biological tissues where they occupy a fundamental position in the intermediate metabolism and in the cells of certain organs, such as the brain and the liver. They are divided into two main classes, namely: the phospholipids (PLS) which give mineral phosphates by hydrolysis, for example phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI); and the glycolipids (GLS) of which the structural bases sphingosine, for example the cerebrosides, hematosides, gangliosides, glycerol, for example the mono- and digalactosyl diglycerides, or sterols, for example the steryl glucosides and their esters, contain at least one sugar residue. The quantity of (GLS) in the CLS is generally 3 to 10 times smaller than that of the PLS.
The animal GLS, which are present in high concentrations in the brain, constitute important functional components of the membranes and are involved in the intramembranal transport, cell recognition, synaptic transmission, cell growth, hormone fixation, enzymes, bacteria, bacterial toxins, malignant tranformations and in many other biological functions. The GLS are thus used, for example, in medicine, in pharmacology, in geriatrics and in cosmetics.
It is known that GLS can be completely or partly extracted from biological tissues by means of various mixtures of polar organic solvents, for example chloroform-methanol, hexane-isopropanol, tetrahydrofuran-water. In these processes, they are only obtained in admixture with the neutral lipids (NLS) and the PLS so that these extracts are called total lipids (TLS). Alternatively, the biological tissues are first extracted with acetone to eliminate a large part of the NLS, for example the triglycerides, sterols and free fatty acids, after which the residual membranal lipids are extracted with the polar organic solvents mentioned above. The TLS may continue to be separated in various ways. The most widely used method is the Folch process which comprises collecting, in a two-phase solvent system of chloroform, methanol and water, an upper phase containing the majority of gangliosides, the GLS containing more than 4 to 5 sugar residues and traces of acidic PLS (APLS) and a lower phase containing the majority of the PLS, the ceramides, the steryl glucosides, the ceramides mono-, di- and trihexosides and all the non-polar lipids, for example the triglycerides. However, the separation of the lipids is not clean so that, for example, hematosides are found in the two phases.
One known large-scale method for separation of the NLS and CLS is based on the use of a mixture of hexane and ethanol. Other methods use liquid chromatography, but are attended by the disadvantage of inadequate capacity, for example 30 mg lipids per g silica gel. A very attractive process which is described in patent application DE 2 915 614 comprises separating the TLS into NLS and PLS on silica gel, although there is no mention of the other CLS, such as the gangliosides or the GLS. In this process, the NLS are defined as that part of the TLS which is soluble in hydrocarbons, as opposed to the PLS which form micelles in those solvents. Only the lipids which form micelles are freely eluted from the silica gel while the NLS remain adsorbed.
However, this process is attended by disadvantages. In fact, many GLS do not spontaneously form micelles in the presence of hydrocarbons. As a result, solid particles insoluble in the hydrocarbons are formed and obstruct the column of silica gel which thus adsorbs the NLS. Accordingly, only the PLS are quantitatively eluted from the column.
Although complete recovery of the GLS from the CL mixtures is possible, it takes an unusually long time. In the standard method, the lipids are first completely dried for 24 to 48 h over phosphorus pentoxide and then acetylated with acetic anhydride in pyridine. The acetylated GLS are then separated from the other lipids by liquid chromatography in a column of Florisil, the GLS are deacetylated and neutralized, the salts are eliminated and, finally, the product is dried by freeze-drying. The method is destructive to all the PLS and to certain GLS containing bonds sensitive to bases, for example the gangliosides with N-O-diacetyl neuraminic acid. In addition, the operation takes several days or even a whole week.
It has been proposed to resolve the difficulties discussed in the foregoing by a simple method of liquid chromatography comprising preparing a reuseable resin containing phenylboronic acid (PBA) groups fixed by covalent bonding to a matrix of crosslinked polystyrene. The resin selectively retains all the GLS by the formation of a complex between the PBA groups and the cis-diol groups of the sugar part of the GLS, thus providing for complete elution of the PLS and NLS. The GLS are then decomplexed with an aqueous organic solvent. However, the charging capacity of the resin is relatively low, typically 0.7 to 7 mg TL per g or 0.18 to 1.8 mg per ml resin (apparent volume). The volume of elution solvent required for 1 mg TL is of the order of 2.5 to 25 ml. It has been found that the commercially available resin cartridges containing 100 mg resin are ineffective for separating the PLS and GLS.